Ataxia-telangiectasia (A-T) is a genetic recessive disorder that affects 1 in 40,000 to 100,000 births. Patients are affected by a large range of symptoms including telangiectasae (dilation of blood vessels) on the eyes, face, and shoulders, ataxia (loss of balance), neurodegeneration, cerebellar degeneration, ocular telangiectasia, radiosensitivity, cancer predisposition, immunodeficiency, and premature aging. A-T cells display cell cycle checkpoint defects, chromosomal instability, and sensitivity to ionizing radiation.
The A-T gene, cloned by positional cloning (Savitsky et al (1995) Hum. Mol. Genet. 4: 2025–2032) encodes a 350 kDa protein kinase known as “ataxia-telangiectasia, mutated” (ATM) involved with the DNA double-stranded break response mechanism and initiation of repair, which are events responsible for maintaining the genomic integrity of the cell. Activation of ATM has effects on multiple signal transduction pathways related to cell cycle checkpoints and DNA damage repair. Complete genomic sequence (184 kb) of the A-T gene, also known as the ATM gene, is disclosed at GenBank Accession No. U82828 (Platzer et al. (1997) Genome Res. 7 (6), 592–605). ATM mRNA is disclosed at GenBank Accession No. U33841 (Savitsky et al (1995) Hum. Mol. Genet. 4: 2025–2032). Cloning, sequences, and organization of the A-T gene are disclosed, inter alia, in U.S. Pat. Nos. 6,265,158, 6,211,336 and 5,858,661 to Shiloh et al., and mutations in the A-T gene are disclosed in U.S. Pat. No. 5,955,279 to Gatti et al.
ATM is a serine/threonine kinase that targets many substrates including p53, RPA, MDM2, NBS1, Chk2, RPA, BRCA1, and other substrates that are postulated but currently unknown. (Gatti et al., (2001) in Metabolic and Molecular Bases of Inherited Disease, 8th Ed, Scriver et al. Eds, pp 705–732) ATM is a member of a family of large kinases containing a C-terminal end homologous to the phosphatidylinositol 3-kinase domain. These proteins play a role in cell cycle checkpoint or DNA damage repair. Other proteins in this family include Rad 3, Mec1p, Mei-41, Rad 50, Tel1 and DNA-PK.
Many aspects of ATM function have been elucidated, but little is known about the structure due to difficulties in isolating ATM. Only a few domains have been identified based on protein homology (Savitsky, K., et al. (1995) Human Molecular Genetics 4: 2025–2032) and biochemical activity (Shafinan, T., et al. (1997) Nature 386: 520–523; Banin, S., et al. (1998) Science 281:1674–1677; Canman, C., et al. (1998) Science 281: 1677–1679).
Overexpression of ATM has been difficult to accomplish due to the instability of the cDNA and the large protein size. Baculovirus expression and protein purification has been attempted (Scott et al. (1998) Biochem Biophys Res Comm 245:144–148) but a high protein yield was difficult to obtain. When ATM was overexpressed in insect cells, only a fraction of recombinant protein was found in the soluble portions of cell preparations, and the majority of the protein was associated with cellular membranes (Ziv et al. (1997) Oncogene 15, 159–167). In 100 ml of infected insect cells, only 20 ng of ATM was produced (Scott et al. (1998) Biochem Biophys Res Comm 245: 144–148), whereas expression of other recombinant proteins often results in recovery of milligram amounts of protein.
Purification of endogenous ATM by conventional biochemical methods has resulted in extremely low yields of purified protein. Smith and colleagues purified ATM from 50 ug of HeLa cell nuclear extract using a series of chromatography columns (Smith et al. (1999) Proc Natl Acad Sci USA 96: 11134–11139). A double-stranded DNA column was used as the last purification step resulting in a homogenous elution. Atomic force microscopy, used to visualize biological interactions, was used to analyze purified ATM and showed that ATM exists as monomers and tetramers. (Smith et al., (1999) Proc Natl Acad Sci USA 96: 11134–11139)
Chan et al. purified endogenous ATM from human placenta using various biochemical chromatographic steps, resulting in approximately 2 ug of ATM protein from 300 grams of placenta tissue, whereas 500 ug of DNA-protein kinase catalytic subunit (DNA-PKcs) protein was isolated from the same tissue. (Chan et al (2000) Jnl Biol Chem 275: 7803–7810) Rhodes et al. purified FLAG-tagged ATM by transiently transfecting an expression construct in HEK 293T cells and isolating ATM using an anti-FLAG affinity column. (Rhodes et al. (2001) Prot Expression and Purif 22: 462–466) Rhodes et al. were able to purify only 1 ug of ATM protein from a 225 cm2 flask that had been seeded with 8×106 uninfected cells and incubated for overnight prior to transfection, and then incubated for another 24 hours after transfection. Thus, the protein recovery reported by Rhodes et al. appeared to be about 1 ug ATM protein from at least 8×106 cells, and relative yield may be even lower if cell division occurred during incubation such that substantially more cells were used for purification. (Rhodes et al. (2001) Prot Expression and Purif 22: 462–466)
A DNA requirement in ATM activation has been reported, but has been disputed. Banin et al and Canman et al. reported ATM kinase activity against p53 substrate, where the activity was independent of DNA. (Banin et al. (1998) Science 281: 1674–1677; Canman et al. (1998) Science 281: 1677–1679) Chan et al. determined that ATM activity and was manganese-dependent and DNA-independent, except when ATM was phosphorylating RPA, in which case DNA was required. (Chan et al (2000) Jnl Biol Chem 275: 7803–7810) Smith et al. used DNA-iron oxide particles as their final purification step to isolate ATM from HeLa cells. (Smith et al. (1999) Proc Natl Acad Sci USA 96: 11134–11139) They reported an increase of kinase activity in the presence of sheared DNA. Using atomic force microscopy, Smith et al. (1999) showed ATM preferentially localizing to ends of DNA double strand gaps, providing some evidence of a protein-DNA interaction. (Smith et al. (1999) Proc Natl Acad Sci USA 96: 11134–11139)